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Comprehensive Characterization of Cells: Techniques and Markers for Accurate Identification

This document outlines the essential methods for characterizing cells, emphasizing various techniques such as species identification and chromosomal analysis. It covers confirmation of species of origin, correlation with tissue types, transformation status, and cross-contamination risks. Key methods include flow cytometry, which allows detection of surface and intracellular markers, and the analysis of unique cell markers. Additionally, practical examples of specific markers for different cell types are provided, highlighting the importance of accurate characterization in biological research and applications.

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Comprehensive Characterization of Cells: Techniques and Markers for Accurate Identification

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  1. Characterizing Cells

  2. What To Characterize • Confirmation Of Species Of Origin • Correlation With The Tissue Of Origin • Transformation Status • Finite Or Continuous • Cross-Contamination • Stability (ex. susceptibility to transformation)

  3. How Is Characterization Done • Species Identification • Chromosomal analysis • Tissue Markers • Cell surface markers • Ex. CD11c if DC • Intermediate filament proteins • Ex. 1 Glial fibrillary acidic protein (GFAP) for astrocytes. Ex. 2 Desmin for muscle cells. Ex. 3 Cytokeratin for epithelial cells • Differentiated products • Ex. Melanin for melanocytes, hemoglogin for erythroid cells, serum albumin for hepatocytes • Unique Markers • Ex. HLA highly polymorphic, unique to an individual • Morphology • Easy and fast but has variability depending on culturing conditions and site

  4. Flow Cytometry Is A Powerful Technique For Characterizing Cells • Allows For Detection Of Surface Markers Of Cells • Allows For Detection Of Intracellular Factors • Allows Detection Of Secreted Factors By Cells • Allows For Detection Of DNA Content

  5. Principles Of Flow Cytometry

  6. How Is It Done: http://biology.berkeley.edu/crl/flow_cytometry_basic.html

  7. Limitations With Light Scattering • Some Information Can Be Obtained • FSC Correlates With Cell Size • SSC Correlates With Internal Complexity • To Distinguish Between 2 Cell types • A. Size Has To Be Different OR • B. Internal Complexity i.e amount of granules • If These Two Parameters Are The Same, Then No Distinction Can Be Made • See The Following Figure

  8. FSC vs SSC Dot Plot

  9. Fluorescence And Antibodies To The Rescue

  10. Fluorescent Dyes And Antibodies • Fluorochromes Are Molecules That Emit Fluorescence Upon Excitation With Light • Ex. FITC (Fluorescein Isothiocyanate) • PE (Phycoerythrin) • PerCP (Peridinin Chlorophyll Protein) • APC (Allophycocyanin) • Some Fluorochromes Are Proteins, Some Are Small Organic Compounds • Ex. PE (Phycoerythrin)-Protein • Ex. FITC (Fluorescein Isothiocyanate)

  11. Principles Of Fluorescence E= h f  = f 

  12. Excitation Spectra Of Fluorochromes

  13. Emission Spectra

  14. Architecture Of A FacsCalibur Instrument

  15. Analyzed Data

  16. GM-CSF+IL-4 GM-CSF+IL-4+PGE2 Fig. 2 GMF: 1.85 GMF: 168.92 25 75 99 1 GMF: 11.82 GMF: 391.83 99 80 1 20

  17. Unstained ISOTYPES

  18. GM-CSF + IL-4 (6 Days Old) GM-CSF

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